Small molecule anticancer compounds and related production process

ABSTRACT

A group of specific branched-chain fatty acids, with significant anticancer effects on human and animals; methods of making using either chemical synthesis or biosynthesis methods; and methods of treating cancer.

This application is a continuation in part of U.S. application Ser. No.09/173,681 filed Oct. 16, 1998, now U.S. Pat. No. 6,214,875, whichapplication claims the benefit of U.S. Provisional Application60/081,712, filed Apr. 14, 1998.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a group of compounds, i.e., specificbranched-chain fatty acids, and pharmaceutically acceptable salts andderivatives thereof, with significant anticancer activities, and methodsof treating cancer. The invention also relates to a process of producingfermentation products containing said specific branched-chain fattyacids, using specific bacteria strains, preferably in industrialfacilities.

2. Description of the Background

Carcinoma is one of the most serious diseases threatening human's healthand life. So far the predominant treatments to cancer patients areradiotherapy and chemotherapy. Both have certain toxicity or sideeffects on humans while suppressing cancer cell growth or killing cancercells. Therefore extensive investigations have been carried out in orderto find an effective anti-carcinogen with minimum side effects andtoxicity.

In 1987, when the inventor cultured K562 leukemia cell lines in thelaboratory, cells in a culture flask were found to have completelydisappeared 48 hours after being contaminated by a kind of rod bacteria.Those rod bacteria were then intentionally harvested and purified, andincubated in soybean media with appropriate inorganic salts. It wasfound in later animal studies that the fermentation solution effectivelyinhibited tumor growth with no toxicity or side effects. In the decadesince then, thousands of cancer patients, including advanced stagecancer patients, have been treated with the oral liquid developed fromthis fermentation solution. These include leukemia, tongue cancer,colorectal cancer, breast cancer, prostate cancer, lung cancer, gastriccancer, hepatocarcinoma, melanocarcinoma, renal cancer, esophagus cancerand pancreas cancer patients. Most of them have responded to the oralliquid, such as by symptom improvement, tumor shrinkage or even completedisappearance. Many of these patients are still alive today. The casesincluded patients in China, Japan, Korea, the United States, and manyother countries.

In order to discover the active components in the fermentation solutionthat play a key role in killing cancer cells, persistent investigationshave been carried out for the last ten years. In this period many booksand papers were published worldwide trying to explain the anticanceractivity of this fermentation solution. Most of these reports suggestedthat some soybean isoflavones (e.g. genistein, daidzein and saponin)from the soybean media contributed to the anticancer activities of thisfermentation solution. On the other hand, some clinical trials indicatedthat the anticancer activities of soybean isoflavones were not greatenough to explain the anticancer effects of the fermentation solution.The inventor has isolated many compounds from the fermentation solutionand revealed that the anticancer activities of the solution were largelycontributed by 13-methyltetradecanoic acid and 12-methyltetradecanoicacid. Further investigations discovered that other members of the familyof branched-chain fatty acids also had significant tumor-inhibitioneffects. So far there are no other reports in the literature regardingthe anticancer activity of specific branched-chain fatty acids.

SUMMARY OF THE INVENTION

The present invention relates to a group of compounds, i.e., specificbranched-chain fatty acids, and pharmaceutically acceptable salts andderivatives thereof, with significant anticancer activities, and methodsof treating cancer using these compounds. Comprehensive biochemical andmorphological tests have demonstrated that these activities areassociated with induction of programmed cancer cell death (apoptosis).Very importantly, the specific branched-chain fatty acids do not killnormal cells. In animal studies, intraperitoneal injection of13-methyltetradecanoic acid daily up to 800 mg/kg to mice did not reachthe LD50 level (50% lethal dose). In human clinical studies, sixvolunteers received 0.6 g–1.8 g 13-methyltetradecanoic acid daily forone month without any side effects.

The specific branched-chain fatty acids can be, but are not limited to,those obtained by synthesis, or by isolation from said fermentationproducts. Particularly, the present invention relates to thefermentation products containing these specific branched-chain fattyacids, which have the capability of inhibiting the growth of cancercells without any toxic or side effects, and the capability of antiagingand immune boosting as well. The present invention also relates to aprocess of producing fermentation products containing the specificbranched-chain fatty acids, using specific bacteria strains, preferablyin industrial facilities.

BRIEF DESCRIPTION OF THE DRAWINGS

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawing(s) will be provided by thePatent and Trademark Office upon request and payment of the necessaryfee.

FIGS. 1A and 1B show the morphological changes of K562 human leukemiacells undergoing apoptosis using transmission electron microscope; A:untreated; B: treated with 13-methyltetradecanoic acid (60 μg/ml) for 4hours.

FIGS. 2A and 2B show the morphological changes of SNU-423 humanhepatocellular carcinoma cells undergoing apoptosis under a lightmicroscope; A: untreated; B: treated with 13-methyltetradecanoic acid(60 μg/ml) for 24 hours.

FIGS. 3A and 3B show the morphological changes of SNU-1 human gastriccarcinoma cell lines stained with H&E under a light microscope; A:untreated; B: treated with 13-methyltetradecanoic acid (60 μg/ml) for 8hours.

FIGS. 4A and 4B show the morphological changes of DU-145 human prostatecarcinoma cell lines stained with H&E under a light microscope; A:untreated; B: treated with 13-methyltetradecanoic acid (60 μg/ml) for 8hours.

FIGS. 5A and 5B show flow cytometric analysis of K562 human leukemiacells; A: untreated; B: treated with 13-methyltetradecanoic acid (30μg/ml) for 24 hours.

FIGS. 6A, 6B and 6C show flow cytometric analysis of MCF-7 human breastadenocarcinoma cells; A: untreated; B: treated with12-methyltetradecanoic acid (60 μg/ml) for 4 hours; C: treated with12-methyltetradecanoic acid (60 μg/ml) for 24 hours.

FIGS. 7A and 7B show flow cytometric analysis of normal human peripheralblood lymphocytes (PBLs); A: untreated; B: treated with13-methyltetradecanoic acid (60 μg/ml) for 24 hours.

FIGS. 8A and 8B show detection of apoptotic SNU-1 cell lines added withTUNEL-(TdT-mediated dUTP nick end labeling) reaction mixture under afluorescence microscope; A: untreated; B: treated with13-methyltetradecanoic acid (60 μg/ml) for 8 hours.

FIGS. 9A, 9B and 9C, show detection of apoptotic K-562 cell lines addedwith peroxidase (POD) and substrate under a light microscope; A:untreated; B: treated with 13-methyltetradecanoic acid (60 μg/ml) for 2hours; C: treated with 13-methyltetradecanoic acid (60 μg/ml) for 4hours.

FIGS. 10A and 10B show detection of apoptotic H1688 cell lines addedwith POD and substrate under a light microscope; A: untreated; B:treated with 13-methyltetradecanoic acid (60 μg/ml) for 8 hours.

FIGS. 11A and 11B show detection of apoptotic DUI45 cell lines addedwith POD and substrate under a light microscope; A: untreated; B:treated with 13-methyltetradecanoic acid (60 μg/ml) for 8 hours.

FIGS. 12A and 12B show normal human PBLs added with POD and substrateunder light microscope; A: untreated; B: treated with13-methyltetradecanoic acid (60 μg/ml) for 8 hours.

FIG. 13 shows DNA fragmentation gel electrophoresis of K562 humanleukemia cells undergoing apoptosis, which were treated with13-methyltetradecanoic acid (60 μg/ml).

FIG. 14 shows caspase target protein Lamin B cleavage in apoptoticSNU-423 human hepatocellular carcinoma cells treated with13-methyltetradecanoic acid (60 μg/ml).

FIG. 15 shows caspase target protein Lamin B cleavage in apoptotic K562human leukemia cells treated with 13-methyltetradecanoic acid (60μg/ml).

FIG. 16 shows caspase target protein Rb hypophoshorylation and cleavagein apoptotic SNU-423 human hepatocellular carcinoma cells treated with13-methyltetradecanoic acid (60 μg/ml).

FIG. 17 shows caspase target protein Rb hypophoshorylation and cleavagein apoptotic K562 human leukemia cells treated with13-methyltetradecanoic acid (60 μg/ml).

FIG. 18 shows comparison of the tumors removed from the mice of twotreated groups and control group of human prostate cancer DU145 nudemice model.

FIG. 19 shows comparison of the tumors removed from the mice of treatedgroup and control group of human hepatocellular carcinoma LCI-D35orthotopic nude mice model.

DETAILED DESCRIPTION OF THE INVENTION

Definitions of the Specific Branched-Chain Fatty Acids

The present invention relates to specific branched-chain saturated andunsaturated fatty acids, with significant anticancer activities, i.e.,terminally methyl-branched iso- and anteiso-fatty acids. The presentinvention also includes any and all derivatives of these fatty acids, solong as the terminally methyl-branched iso- or anteiso-fatty moietyremains. These fatty acids can be characterized by the formula R₀COOH,wherein R₀ represents a terminally methyl-branched iso- or anteiso-fattygroup. By the term “terminally methyl-branched iso” and “terminallymethyl-branched anteiso”, it is intended that the end of the R₀ groupfarthest away from the COOH group have the following formulae,respectively:

The portion of the fatty group R₀ other than the terminally-methylbranched iso- or anteiso-moiety, as described above, is not limited andmay be saturated or unsaturated, linear or branched, for example.

An embodiment of the methyl-branched saturated fatty acids wherein theabove portion of the fatty group R₀ other than the terminally-methylbranched iso- or anteiso-moiety is linear can be described by theformula (I):

In the above formula (I), m is 0 or 1, and n is an integer. There is nolower or upper limit for n so long as the acid is a fatty acid. Thus,n+m may range as high as 96 or higher, with an upper limit of 46 beingpreferable. More preferably, n is 7–16.

The methyl-branched unsaturated fatty acids have the above formula,except that n is at least 2, and at least one CH₂—CH₂ group in (CH₂)_(n)is replaced with a CH═CH group.

The terminally methyl-branched iso-fatty acids are the methyl-branchedsaturated fatty acids having x carbons and n=x−4, m=0 in the aboveformula, and known as “iso-Cx” in the present invention. For example,13-methyltetradecanoic acid is expressed as “iso-C15” and has theformula

The terminally methyl-branched anteiso-fatty acids are themethyl-branched saturated fatty acids having x carbons and n=x−5, m=1 inthe above formula, and known as “anteiso-Cx” in the present invention.For example, 12-methyltetradecanoic acid is expressed as “anteiso-C15”and has the formula

An example of a terminally methyl-branched unsaturated iso-fatty acid ofthe present invention is

otherwise known as iso-17:1 ω9c.

The present invention also includes pharmaceutically acceptable salts ofsaid terminally methyl-branched iso- and anteiso-fatty acids, which areobtained by reaction with inorganic bases, such as sodium hydroxide, andhave the ability to inhibit cancer cell growth. Such compounds includeR₀COONa having not less than 12 carbons and R₀COOK having not less than6 carbons, wherein R₀ is as defined above, Na is sodium, and K ispotassium.

The present invention also includes pharmaceutically acceptablelipoproteins of said terminally methyl-branched iso- and anteiso-fattyacids, which are obtained by conjugation with proteins, includingpolypeptides and oligopeptides, and have the ability to inhibit cancercell growth. Such lipoproteins are well known in the art.

The present invention also includes all pharmaceutically acceptablederivatives other than lipoproteins, such as amides, esters, etc., ofsaid terminally methyl-branched iso- and anteiso-fatty acids, which areobtained by reaction of the fatty acid with the corresponding amine,alcohol, etc. precursor, and have the ability to inhibit cancer cellgrowth. Such derivatives include, but are not limited to, those thathave the formula R₀CO-A, where R₀ is as previously defined, and Arepresents one of the following groups:

In the above formula 7, R₀′ has the same definition as R₀ but may be thesame or different. In the above formula 13, R is a side chain of anamino acid, and n is 0 or an integer.

The present invention also includes said terminally methyl-branched iso-and anteiso-fatty acids, wherein one or both hydrogens in a —CH₂-groupis substituted with a group X, such as Cl, I, Br, OH or NH₂, and havethe ability to inhibit cancer cell growth. Examples of such substitutedfatty acids have the formula R₀CHXCOOH or R₀CX₂COOH, and more than 8carbon atoms, wherein R₀ is as defined above. Such compounds include:

and

The present invention also includes pharmaceutically acceptable salts,lipoproteins, and other derivatives of the above substituted fattyacids.

The terminally methyl-branched iso- and anteiso-fatty acids of thepresent invention can be obtained by, but not limited to, isolation fromfermentation or incubation products using specific bacteria, or bychemical synthesis, or by extraction from natural materials.

Having generally described this invention, a further understanding canbe obtained by reference to certain specific examples which are providedherein for purposes of illustration only and are not intended to belimiting unless otherwise specified.

I. Demonstration of Anticancer Activity and Safety of SpecificBranched-Chain Fatty Acids

EXAMPLE 1 Anticancer Activity In Vitro

Samples:

iso-C15, including extracted and synthesized.

The extracted iso-C15 was isolated by HPLC (High Performance LiquidChromatography) from the fermented solution (fermented using thespecific bacteria, Stenotrophomonas maltophilia Q-can, and media andproduction process in present invention).

The synthesized iso-C15 was purchased from Sigma Chemical Company (St.Louis, MO.)

-   The other specific branched-chain fatty acids tested include:-   10-methylundecanoic acid (iso-C12),-   11-methyllauric acid (iso-C13),-   12-methyltridecanoic acid (iso-C14),-   11-methyltridecanoic acid (anteiso-C14),-   12-methyltetradecanoic acid (anteiso-C15),-   14-methylpentadecanoic acid (iso-C16),-   13-methylpentadecanoic acid (anteiso-C16),-   15-methylpalmitic acid (iso-C17),-   16-methylheptadecanoic acid (iso-C18),-   15-methylheptadecanoic acid (anteiso-C18),-   17-methylstearic acid (iso-C19),-   18-methylnonadecanoic acid (iso-C20).

All the samples above were purchased from Sigma Chemical Company.

Cell Lines:

Human leukemia cell line K562 and human gastric cancer cell lineSGC7901.

Methods:

MTT assay was performed to test the cytotoxicity. The K562 and SGC7901cells were maintained in exponential growth in RPMI 1640 mediumsupplemented with 15% heat-inactivated newborn calf serum. The cellswere plated at a density of 2×10⁴ cells/100 μl medium/well into 96-wellplate with medium containing samples in five final concentrations (7.5,15, 30, 60 and 90 μg/ml) for iso-C15 (either synthesized or extracted)and one final concentration (30 μg/ml) for the others. The media incontrol wells contained no samples. The cells were incubated at 37° C.in a highly humidified incubator under 5% CO₂ atmosphere for 24 hours.The supernatant was removed by fast inversion of the plate. 20 μl of 5mg/ml MTT solution were added into each well. Incubation was continuedfor 4 hours. DMSO 100 μl/well was added and the plate was vibrated for10 minutes. A_(570nm) was read at the Immunoreader BioTek EL311S.

The inhibition rate (%)=1−(mean A_(570nm) in test wells/mean A_(570nm)in control wells)

Results:

TABLE 1 Inhibitory rate (%) of synthesized iso-C15* on cell growth Cellline 90 μg/ml 60 μg/ml 30 μg/ml 15 μg/ml 7.5 μg/ml K562 85.3 83.1 71.650.1 26.2 SGC7901 68.4 63.1 50.5 27.5 — *the sample was dissolved with10% ethanol.

TABLE 2 Inhibitory rate (%) of extracted iso-C15* on cell growth Cellline 90 μg/ml 60 μg/ml 30 μg/ml 15 μg/ml 7.5 μg/ml K562 87.2 83.7 72.251.2 27.1 SGC7901 68.8 62.1 51.2 28.1 — *the sample was dissolved with10% ethanol.

TABLE 3 Inhibitory rate (%) of specific branched-chain fatty acids* onK562 cell growth Sample iso-C12 iso-C13 iso-C14 iso-C16 iso-C17 iso-C18% 70.69 71.03 72.15 71.58 70.79 68.39 anteiso- anteiso- anteiso- Sampleiso-C19 iso-C20 C15 C14 C16 anteiso-C18 % 69.15 62.58 73.10 72.59 70.6871.73 *the concentration of branched-chain fatty acids was 30 μg/ml; thesample was dissolved with NaOH solution to adjust to pH 7.5.

EXAMPLE 2 Determination of ID₅₀, ID₇₅ and ID₉₀

Samples:

The extracted iso-C15 was isolated by HPLC from the fermented solution(fermented using the specific bacteria, Stenotrophomonas maltophiliaQ-can, and process of the present invention, infra). The samples wereprepared by dissolving them in NaOH solution (adjusted to pH7.5) and0.5% Tween 80 (Sigma Chemical Company, St. Louis, Mo.).

Cell Lines:

All tumor cell lines were purchased from American Type CultureCollection (ATCC, Manassas, VG) and were cultured as recommended byvendor. Human PBLs were separated from whole blood of healthyindividuals by using Ficoll-Hypaque gradients. They were maintained insuspension in RPMI 1640 with 10% plasma from the same individuals. Allcell cultures were incubated in a CO₂ atmosphere (5%) at 37° C.

Seven human tumor cell lines were studied. K-562 human leukemia andSNU-1 human gastric carcinoma cell lines were cultured in suspension inRPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum(FBS). MCF-7 human breast adenocarcinoma, DU-145 human prostatecarcinoma, SNU-423 human hepatocellular carcinoma, HCT 116 human coloncarcinoma, and H1688 human small cell lung carcinoma cell lines werepropagated as adherent cells in RPMI 1640 supplemented with 10%heat-inactivated FB S (for SNU-423 and H1688), or in McCoy's 5a mediumwith 10% heat-inactivated FBS (for HCT 116), or in minimum Eagle'smedium with 10% heat-inactivated FBS (for MCF-7 and DU-145).

Methods:

All cells in adherent culture were initiated at 5×10⁴ cells/well in96-well microplates and treated immediately with iso-C15 at differentconcentrations (0, 1.5, 3.0, 6.0, 15.0, 30.0, and 60.0 μg/ml) dilutedwith medium. Both untreated and solvent (NaOH and Tween 80) treatedcells served as controls. The treated cells were incubated for 48 hoursat 37° C. After incubation, the supernatants were removed and the cellswere trypsinized and collected prior to viability assessment by trypanblue dye exclusion.

PBLs, K-562 and SNU-1 cells in suspension culture were seeded in 96-wellmicroplates at a density of 5×10⁴ cells/well for K-562 and SNU-1, and1×10⁵ cells/well for PBLs. iso-C15 were diluted with medium to providedifferent concentrations (0, 1.5, 3.0, 6.0, 15.0, 30.0, and 60.0 μg/ml).Both untreated and solvent (NaOH and Tween 80) treated cells served ascontrols. After incubation for 48 hours at 37° C., cells were collecteddirectly from the wells for viability assessment.

The ID₅₀, ID₇₅ and ID₉₀ were determined in duplicate in every set ofexperiments, and each experiment was repeated three times underidentical conditions. ID₅₀, ID₇₅ and ID₉₀ were defined as theconcentration of iso-C15 required to kill 50, 75 or 90%, respectively,of cells (compared with that in untreated cells) and computed usingCalcuSyn for Windows software (Biosoft, Cambridge UK) based on MedianEffect method by Dr. T. C. Chou.

Results:

The cytotoxic activity of iso-C15 was quantified by determining ID₅₀,ID₇₅ and ID₉₀ (μg/ml or μM) in several human hematological and solidtumor cell lines. It is indicated from Table 4 that iso-C15 was activein all tumor cell lines studied. The strongest cytotoxic activities werefor MCF-7 human breast adenocarcinoma and K-562 human leukemia. Theactivities were less for H1688 human small cell lung carcinoma and HCT116 human colon carcinoma cell lines. In contrast, iso-C15 is not toxicagainst normal human peripheral blood lymphocytes at concentrationslethal to tumor cells.

TABLE 4 Cytotoxicity of iso-C15 on human tumor and normal cells in vitrocell line cell type ID₅₀(μg/ml) ID₇₅(μg/ml) ID₉₀(μg/ml) MCF-7 breastcarcinoma 10.03 ± 0.97 15.99 ± 1.28 25.49 ± 1.68 K-562 leukemia 11.45 ±1.82 22.27 ± 4.60 43.57 ± 6.71 DU145 prostate carcinoma 13.98 ± 2.1540.43 ± 5.72 81.87 ± 8.85 H1688 lung carcinoma 15.08 ± 1.92 35.03 ± 3.5961.37 ± 8.06 HCT-116 colon carcinoma 18.49 ± 6.23 67.96 ± 8.25 108.65 ±13.35 SNU-1 gastric carcinoma 20.77 ± 2.47 47.43 ± 4.95  80.49 ± 10.03SNU-423 hepatocarcinoma 24.26 ± 3.98 70.46 ± 9.36 120.77 ± 15.82 PBLnormal human lymphocytes >400

EXAMPLE 3 In Vitro Introduction of Apoptosis in Human Tumor Cell Linesand Molecular Pathway

Reagents:

RPMI 1640, DMEM and McCoy culture medium, as well as Fetal and calfbovine serums were purchased from Life Technologies (Long Island, N.Y.).Argarose for DNA gel electrophoresis was purchased from FMC, andAcrylamide for Western blot was from Bio-Rad. Antibodies against humanc-myc, caspase 3, caspase 8, poly (ADP-ribose) polymerase (PARP),lamins, p53 and retinoblastoma (Rb) were from Oncogene. Chemicals usedin buffers and other reagents were from Sigma (St. Louis, Mo.).

13-methyltetradecanoic acid (iso-C15) was chemically synthesized in theinventor's laboratory, as described in Example 5, infra, (purity of99.8%) and 12-methyltetradecanoic acid (anteiso-C15) purchased fromSigma were prepared by dissolving in NaOH solution and then in 0.5%Tween 80 with pH7.5.

Cell Culture:

Human cancer cell lines DU-145 (prostate cancer), K562 (leukemia),HCT116 (colon cancer), H1688 (lung cancer), SUN423 (hepatocarcinoma),MCF7 (breast cancer), CRL-1687 (pancreatic cancer), and SUN-1 (gastriccancer) were obtained from American Type Culture Collection (ATCC). 30ml blood was collected from a health person and normal peripheralmononuclear cells were separated by Ficoll separation solution (Sigma).All cells were maintained in RPMI 1640, DMEM, or McCoy mediumsupplemented with 10% FCS, 100 mg/ml streptomycin and 100 u/mlpenicillin. Normal human peripheral mononuclear cells, K562 and SUN-1were suspended cells. After spinning at 1,500 RPM for 5 min,supernatants were discharged and cells were resuspended and expended infresh medium. The other tumor cell lines were adherent cells and weredispersed with 0.05% trypsin/0.01% EDTA (Irvine Scientific, CA) forexpansion. Cells were seeded in T75 flasks at 2×10⁶ cells/flask inculture medium supplemented with 10% fetal bovine serum and incubatedovernight at 37° C. with 5% CO₂. The adherent cells attached to theplate were striped with disposable cell scrapers (Fisher Scientific)after treated either with 1% iso-C15, anteiso-C15 or control solutionfor 1, 2, 4, 8 and 24 hours and then combined with respective floatcells. Cells were then prepared for flow cytometry analysis, in situcell death detection, DNA fragmentation and Western blot assay followedthe preparation methods for each assay. Cell pellets treated for 2 and 4hr with either iso-C15 or control were also stored at −70° C. for futurestudies of gene regulation.

Methods:

The apoptosis (programmed cell death) of cancer cells induced byspecific branched-chain fatty acids was confirmed by: (a) morphology,visualizing morphological changes indicative of apoptosis; (b) flowcytometry, identifying the cells undergoing apoptosis and discriminatingapoptosis from necrosis; (c) in situ cell death detection kit, POD,detecting apoptosis induced DNA strand breaks at single cell level; (d)gel electrophoresis assay, visualizing apoptotic DNA fragmentation.

The molecular mechanism of apoptosis induced by specific branched-chainfatty acids was studied using Western blot analysis.

A flow cytometer (FACScan) with Consort 30 software for gating analysis(Becton Dickinson, San Jose, Calif.) was used. The Apoptosis Detectionkit (R&D Systems) was used to quantitatively determine the percentage ofcells undergoing apoptosis by virtue of their ability to bind annexin Vand exclude propidium iodide (PI). Cells were washed in cold PBS twiceand resuspended in binding buffer. Fluorescent-labeled annexin V and PIwere added to the cells. The cells undergoing apoptosis, expressingphosphotidyiserine on the outer leaflet of cell membranes, would bindannexin V. The cells in later stage of apoptosis or necrosis, with acompromised cell membrane, would allow PI to bind to the cellular DNA.The resulting cells were immediately analyzed by flow cytometer equippedwith a single laser emitting excitation light at 488 nm. The annexin Vand PI generated signals can be detected in signal detector FL1 and FL2,respectively. Three potential populations of cells can be presented inFL1/FL2 pattern: live cells would not stain with either fluorochrome(zone 3), necrotic and later apoptotic cells would stain with bothfluorochromes (zone 2) while cells undergoing apoptosis would stain onlywith annexin V (zone 4).

In Situ Cell Death Detection Kit, POD (Mannheim Boehringer GmbH) wasused to detect the individual apoptotic cells. Cleavage of genomic DNAduring apoptosis may yield double-stranded, low molecular weight DNAfragments as well as single strand breaks in high molecular weight DNA.Those DNA strand breaks can be identified by labeling free 3′-OH terminiwith modified nucleotides in an enzymatic reaction. In this kit terminaldeoxynucleotidyl transferase (TdT) is used to label free 3′-OH ends ingenomic DNA with fluorescein-dUTP. The incorporated fluorescein isvisualized under fluorescence microscope directly. The incorporatedfluorescein can also bind to anti-fluorescein antibody POD and bedetected by substrate reaction. Stained cells can be analyzed underlight microscope.

The gel electrophoresis assay was used for the detection ofapoptosis-specific internucleosonal DNA degradation in these cells.Tumor cell pellets, treated with 1% iso-C15 and controls, were lysed in1 ml hypotonic lysis buffer (10 mM Tris, pH 7.5, 1 mM EDTA, and 0.2%Triton x-100). After centrifuged at 14,000 RPM for 20 min at 4° C., thesupernatants were transferred to new tubes and treated with RNase andproteinase K respectively. Supernatants were extracted withphenol/chloroform twice, and fragmented DNA was precipitated in ethanol.Samples were electrophoresed in a 1.5% agarose gel in 1× TAE buffer. Thegel was stained with ethidium bromide and destained with distilledwater. The fragmented DNA was then visualized under UV light.

For Western blot assay, each cell pellet collected from 1% iso-C15 orcontrol treated cultures was lysed in 150 μl lysis buffer with 0.5%NP-40, 0.5% deoxycholic acid and 1 mM PMSF. The cell lysates were mixedwith equal volume 2× Laemmli buffer and boiled for 5 min before loadedinto gel wells. Proteins were resolved in an 8% SDS-PAGE gel andtransferred to nitrocellulose filter membrane. The filters were blockedwith PBS-T (PBS with 0.1% Tween 20) containing 5% nonfat dry milk(Bio-Rad, Richmond, Calif.) for 1 hr and then incubated for 1 hr withproper dilution of one primary antibody in PBS-T containing 2% nonfatdry milk. The filters then were washed in PBS-T 5 min for 6 times andincubated with a 1:8000 dilution of HRP secondary antibody in PBS-T with2% nonfat dry milk for 1 hr. After 6 washes in PBS-T, immune complexeswere visualized on film using the ECL nonradioactive detection system(Amersham, Arlington Heights, Ill.). After detected with one primaryantibody, the filter was striped with 0.1 mM Tris pH 7.5 and 0.05 mMβ-metacapenanol at 50° C. for 30 min. The filters were washed in 300 mlPBS-T buffer for 10 min twice before blocking with PBS-T with 5% nonfatdry milk. The membranes were then reprobed with monoclonal mouseanti-human β-actin to determine the equal loading of protein for eachwell.

Results:

Morphological Changes:

The apoptosis of cancer cells is morphologically characterized by cellshrinkage, chromatin condensation, nuclear fragmentation, intact cellmembrane and extensive formation of membrane blebs and apoptotic bodies.

FIG. 1 shows the morphological changes of K562 leukemia cells undergoingapoptosis using transmission electron microscope. Comparing to theuntreated intact cell (FIG. 1A), the cell treated with13-methyltetradecanoic acid (60 μg/ml) for 4 hours (FIG. 1B) exhibitstypical apoptotic feature, chromatin condensed into dense masses againstthe nuclear membrane, membrane intact and cell shrinkage.

FIGS. 2–4 illustrate the morphological changes of cancer cellsundergoing apoptosis in a light microscope. Cultured SNU-423 humanhepatocellular carcinoma cells treated with anteiso-C15 (60 μg/ml) for24 hours (FIG. 2B) exhibited cell volume decrease due to shrinkage andbubbles inside the cell, compared to untreated control (FIG. 2A).Cultured SNU-1 human gastric carcinoma cell lines were treated withanteiso-C15 (60 μg/ml) for 8 hours, and cellular morphology wasevaluated in preparations stained with H&E (FIG. 3B). Compared tountreated control (FIG. 3A), chromatin condensation and cytoplasmicgranularity were noted. Cultured DU-145 human prostate carcinoma celllines were treated with iso-C15 (60 μg/ml) for 8 hours, and cellularmorphology was evaluated in preparations stained with H&E dye (FIG. 4B).Compared to untreated control (FIG. 4A), membrane blebs were noted.

Flow Cytometry:

At least 10⁴ cell events were analyzed. The FL1/FL2 pattern of untreatedK562 human leukemia cells (FIG. 5A) revealed the expected distributionof cells in zone 3. After treatment of K562 cells with iso-C15 (30μg/ml) for 24 hours (FIG. 5B), the majority of the cells were undergoingapoptosis (zone 4, Annexin V positive and PI negative). The kineticbehavior of anteiso-C15 in MCF-7 human breast adenocarcinoma cells wasevidenced by FIGS. 6A, 6B and 6C, for treatment of MCF-7 cells withanteiso-C15 (60 μg/ml) for 0, 4 and 24 hours, respectively. Aftertreatment of anteiso-C15 for 4 hours, many cells were undergoingapoptosis (zone 4, FIG. 6B), while after 24 hours the majority of cellshad already died (later stage of apoptosis, zone 2, FIG. 6C). The flowcytometric analysis of untreated normal human PBLs (FIG. 7A) and treatedPBLs with iso-C15 (60 μg/ml) for 24 hours (FIG. 7B) resulted in nearlyidentical FL1/FL2 patterns (zone 3, viable and not undergoingapoptosis), revealing no significant effects by iso-C15 on normal humanlymphocytes.

In Situ Cell Death Detection:

Four human tumor cell lines, K-562 human leukemia, SNU-1 human gastriccarcinoma cell lines, MCF-7 human breast adenocarcinoma and H1688 humansmall cell lung carcinoma cell lines, as well as Human PBLs were treatedwith iso-C15 (60 μg/ml).

SNU-1 cells treated with iso-C15 for 8 hours were added withTUNEL-reaction mixture and incubated 60 min at 37° C. After washing withPBS for three times, cell morphology was analyzed directly underfluorescence microscopy. Several yellow fluorescent spots of apoptoticcells were noted in cells treated for 8 hours (FIG. 8B), comparing tountreated ones (FIG. 8A).

H1688, K-562 and DU145 human cancer cells and normal human PBLs wereadded with POD and incubated 30 min at 37° C., washed three times withPBS, then reacted with substrate AEC and incubated for 10 min at roomtemperature. The cells were analyzed under light microscope. ComparingK-562 leukemia cells untreated (FIG. 9A) and treated for 2 and 4 hours(FIGS. 9B and 9C), it is found that some cells started apoptosis(stained red) 2 hours after treatment and the number of apoptotic cellsincreased with exposure time. The apoptotic H1688 cancer cells (stainedred) were found after 8 hours of treatment (FIG. 10B) comparing tountreated (FIG. 10A). Some stained apoptotic DU145 cancer cells wereshown 8 hours after treatment (FIG. 11B) and no stained cells inuntreated control (FIG. 11A). In contrast, untreated and 8-hour treatedPBLs were almost the same (FIGS. 12A and 12B), and few stained apoptoticcells were seen. It is evidenced that iso-C15 induces apoptosis ofcancer cells but not normal human cells.

DNA Fragmentation Gel Electrophoresis:

DNA fragmentation electrophoresis is one of most common applied methodsto illustrate the apoptotic changes in experimental cells. Results forK562 leukemia cell line treated with iso-C15 (60 μg/ml) were shown inFIG. 13. The lane of control treated for 8 hours showed only DNA smear.The fragmented low molecular weight DNA bands were seen at 2 hour andwere prominent at 8 hour treated. The appearance of an oligonucleosomalladder in treated cells indicated the break of double-stranded DNA dueto apoptosis induced by iso-C15.

Western Blot Analysis:

The Western blot analysis results (FIGS. 14–17) are used as examples toreveal the signal transduction pathway for specific branched-chain fattyacid to activate apoptosis of cancer cells.

The cleavages of Lamin B, a caspase target protein, in apoptotic SNU-423human hepatocellular carcinoma cells and K562 human leukemia cells wereshown in FIGS. 14 and 15, respectively. The cells were treated with 1%control solution and 1% iso-C15 for the length of time indicated. Celllysates were separated by SDS-PAGE. Lamin B was detected byimmunoblotting with a monoclonal antibody. The cleaved 45 kDa and 32 kDaproducts were shown in FIG. 14, and the cleaved 45 kDa products in FIG.15. The cleavage of caspase target protein Lamin B suggested theactivation of the caspase cascade during apoptosis. The Western blotanalysis of RB protein in SNU-423 and K562 cells were shown in FIGS. 16and 17, respectively. The results showed that iso-C15 induced the changeof hyperphosphorylated RB (pRB120/hyper) to hypophosphorylated form(pRB115/hypo), and also induced the cleavage of full length RB to pRB68kDa fragment in FIGS. 16 and 17, and even smaller pRB48 kDa fragment inFIG. 16.

EXAMPLE 4 Anticancer Activity In Vivo

A. Determination of LD₅₀

Materials and Methods:

13-methyltetradecanoic acid (iso-C15) purchased from Sigma (St. Louis,Mo.) was prepared by dissolving in NaOH solution and then in 0.35% Tween80 with pH7.5.

ICR mice weighing 20.5–22.5 g of both sexes were treated with iso-C15i.p. qd×3 in test groups and with solvent of same dose as in a controlgroup. The doses ranged from 10 to 800 mg/kg of iso-C15 and two micewere included in each dose group (10 mg/kg, 20 mg/kg, 40 mg/kg, 80mg/kg, 160 mg/kg, and 800 mg/kg). The general condition of these micewere monitored daily for seven days.

Results:

No mice died after seven-day administration of iso-C15 of dose up to 800mg/kg. It is shown that iso-C15 is basically not toxic to mice and 50%lethal dose (LD₅₀) was not determined.

B. Efficacy Evaluation of Iso-C15 in Orthotopic Nude Mice Model of HumanProstate Carcinoma DU145

Material and Methods:

13-methyltetradecanoic acid (iso-C15) was chemically synthesized in theinventor's laboratory, as described in Example 5, infra, (purity of99.8%) was prepared by dissolving in NaOH solution and then in 0.35%Tween 80 with pH7.5.

Total of 24 male athymic BALB/c nude mice between 4 and 5 weeks of agewere bred and maintained in specific pathogen free condition.

Human prostate carcinoma DU145 tumor was implanted and maintainedsubcutaneously in the flank of athymic nude mice. Prior to orthotopicimplantation, the tumor was harvested in log phase. The peripheral tumortissue was collected and minced to small pieces of one cubic millimetereach.

The mice were anesthetized for surgical orthotopic implantation. A smallincision was made along the midline of the lower abdomen. After properexposure of the bladder and prostate, the capsule of the prostate wasopened and three pieces of DU145 tumor fragments were inserted into thecapsule. The capsule was then closed using 8-0 suture, and the abdomenwas closed using a 6-0 surgical suture.

The mice bearing orthotopic DU145 were randomly divided into control andtest groups of eight mice each at the second day after tumorimplantation. The iso-C15 prepared above at doses 35 and 70 mg/kg andPBS were administered by gavage once a day in low dose and high dosetest groups and control group, respectively, for 43 days.

All the mice were sacrificed by CO₂ inhalation at day-40 after the startof treatment. The weights of primary tumors and bodies were measured.Tissue samples of the primary tumors were processed through standardprocedures of hematoxylin and eosin staining for microscopicexamination.

The tumor inhibition rates (TIR) were determined by comparing the meantumor weight of the test groups (T) with that of the control group (C)and expressed as a (C)−T)/C percentage, and were analyzed by Student'stest for statistical significance.

Results:

Very promising antitumor efficacy was observed for iso-C15 at doses 35mg/kg and 70 mg/kg in this nude mouse model of human prostate carcinomaDU145 with the tumor inhibition rates 54.8% (p<0.05) and 84.6% (p<0.01)as shown in Table 5.

TABLE 5 Efficacy of iso-C15 on primary tumor and body weight in nudemouse model of human prostate carcinoma DU145 No. of Mean tumor GroupRoute mice weight (mg) TIR (%) P Control Oral 8 1,090.75 — — Low dose,35 mg/kg Oral 8 493.25 54.8 0.042 High dose, 70 mg/kg Oral 8 168.00 84.60.007

For comparison, all the primary tumors after removal from the nude miceare shown in FIG. 18, where it is noted that the implanted tumor did notgrow in four mice in the high dose treatment group. There are no signsof toxicity, as judged by the body weight curve and histology slides.

C. Efficacy Evaluation of Iso-C15 in Orthotopic Nude Mice Model of HumanHepatocellular Carcinoma LCI-D35

Material and Methods:

13-methyltetradecanoic acid (iso-C15) was chemically synthesized in theinventor's laboratory, as described in Example 5, infra, (purity of99.8%), was prepared by dissolving in NaOH solution and then in 0.35%Tween 80 with pH7.5.

Total of 16 male and female athymic BALB/c nude mice between 4 and 5weeks of age were bred and maintained in specific pathogen freecondition.

Human hepatocellular carcinoma LCI-D35 was originally obtained from theprimary tumor of a 45-year-old female patient. The tumor was implantedand maintained subcutaneously in athymic nude mice. Prior to orthotopicimplantation, the tumor was harvested in log phase. The peripheral tumortissue was collected and minced to small pieces of one cubic millimetereach.

The mice were anesthetized for surgical orthotopic implantation. A smallincision was made along the midline of the upper abdomen. The left lobeof the liver was exposed and a small incision was made on the liversurface. Two of the tumor fragments above were sutured into the incisionusing 8-0 suture. The abdomen was then closed using a 6-0 surgicalsuture.

The mice bearing orthotopic LCI-D35 were randomly divided into controland test groups of eight mice each at the second day after tumorimplantation. The iso-C15 prepared above at dose 70 mg/kg and PBS wereadministered by gavage once a day in the test and control group,respectively, for 40 days.

All the mice were sacrificed by CO₂ inhalation at day-40 after the startof treatment. The weights of primary tumors and bodies were measured.Tissue samples of the primary tumors were processed through standardprocedures of hematoxylin and eosin staining for microscopicexamination.

The tumor inhibition rates (TIR) were determined by comparing the meantumor weight of the test groups (T) with that of the control group (C)and expressed as a (C)−T)/C percentage, and were analyzed by Student'stest for statistical significance.

Results:

Very promising antitumor efficacy was observed for iso-C15 at dose 70mg/kg in this nude mouse model of human hepatocellular carcinoma LCI-D35with a tumor inhibition rate 64.9% (p<0.01), as shown in Table 6.

TABLE 6 Efficacy of iso-C15 on primary tumor and body weight in nudemouse model of human hepatocellular carcinoma LCI-D35 mice No. bodyweight tumor weight TIR group dose route in./fi. in./fi. mean ± SD (g)(%) p PBS — oral 8/8 17.31/21.50 0.202 ± 0.117 — iso-C15 70 mg/kg oral8/8 18.23/21.75 0.071 ± 0.052 64.9 0.0086

For comparison, all the primary tumors after removal from the nude miceare shown in FIG. 19, where it is noted that the implanted tumor did notgrow in two mice in the treatment group. There are no signs of toxicity,as judged by the body weight curve and histology slides.

D. Human Clinical Studies on the Safety of Iso-C15

Material and Method:

Chemically synthesized iso-C15 of 99.8% purity were prepared in 0.20 gcapsules. Six healthy adult volunteers (4 male, 2 female) of average age35.6 were divided into three groups, and orally received iso-C15capsules for thirty days. Low dose group: one case, 0.6 g daily; middledose group: two cases, 1.2 g daily; high dose group: three cases, 1.8 gdaily.

The examinations were carried out before, during and after experiment,including physical examinations, blood and urine routine examinations,function of heart, liver and kidney, X-ray radioscopy of lung andsubjective symptom.

Results:

The effects of iso-C15 on blood routine and platelet shown in Table 7indicated significant increase of white blood cells (WBC) andgranulocyte (GRAN), while no significant changes of red blood cells(RBC), hemoglobin (HB) and platelet (PLT). No abnormality was observedfrom alanine aminotransferase (ALT) and blood urea nitrogen (BUN) forall subjects, as shown in Table 8. No abnormality was observed on heartand lung from electrocardiogram (EKG) and X-ray radioscopy, and noabnormality on urine routine as well.

TABLE 7 Effects of iso-C15 on blood routine and platelet X ± SD ItemCase Before test After test p WBC 6  5.80 ± 0.95 × 10⁹/L 7.50 ± 0.710.0072 GRAN 6  3.20 ± 0.59 × 10⁹ 3.80 ± 0.89 0.0311 RBC 6  4.64 ± 0.40 ×10¹²/L 4.62 ± 0.69 0.8719 HB 6 122.00 ± 10.40/L 128.00 ± 16.90  0.1839PLT 6  203.0 ± 33.3 × 10⁹/L 232.0 ± 52.1  0.0809

TABLE 8 Effects of Iso-C15 on Liver And Kidney Functions X ± SD ItemCase Before Test After Test p ALT 6 15.17 ± 8.70 μ/L 15.00 ± 10.330.4398 BUN 6  6.69 ± 2.14 mmol/L  6.47 ± 1.72 0.02850

It indicated a wide safety range of 13-methyltetradecanoic acid for oraladministration (0.6 g–1.8 g daily for one month) without any toxicityeffects. It also showed an increase of white blood cells andgranulocytes, indicating an immune boosting effect.

The branched-chain fatty acids can be administered in the form ofliquid, powder, tablet, capsule, injection or encapsulated withliposome, to be delivered by bypassing the digestive tract, or directlyinto the bloodstream. They also can be used as a topical drug for skincancer or other skin diseases.

E. Chemoprevention of 7,12-Dimethylbenz(a)-Anthracene (DMBA)-InducedMammary Carcinogenesis in Rats by Iso-C15

Materials and Method:

13-methyltetradecanoic acid (iso-C15) was prepared by dissolving in NaOHsolution and then in 0.35% Tween 80 with pH 7.5.

Fifty female Sprague-Dawley rats were maintained on laboratory chow. At50 days of age, rats were given a single dose (5 mg) of DMBA (SigmaChemical Co., St. Louis. Mo.) via an intragastric tube. Seven days afterDMBA administration, the rats were randomly divided into two groups of25. Each rat in the control group was given 0.5 ml of 0.35% Tween 80five times a week via an intragastric tube throughout the experiment,while each in the test group was given 0.5 ml iso-C15 solution ofconcentration 0.7%, five times a week. Food and water were available adlibitum. Twenty weeks after DMBA administration, all rats werescarified, and all palpable tumors were removed.

Results:

TABLE 9 Effects of iso-C15 on tumorigenesis of DMBA-induced mammarycancer in rats No. of No. of rats tumor No. of tumors No. of tumors/mean tumor Group Case with tumors incidence tumors per rat tumor bearingrat weight (g) control 25 21 84% 52 2.1 ± 1.7 2.5 ± 2.4 2.4 ± 5.8iso-C15 25 3 12% 4 0.2 ± 0.1 1.3 ± 0.5 0.8 ± 1.3

It is shown in Table 9 that iso-C15 significantly reduced the incidenceof mammary cancer. Iso-C15 has offered prevention in experimentalmammary carcinogenesis. It caused slower tumor growth, though it couldnot achieve a complete prevention.

F. Chemoprevention of Ultraviolet B Ray (VB)-Induced Skin Cancer byIso-C15

Materials and Method:

13-methyltetradecanoic acid (iso-C15) was prepared by dissolving in NaOHsolution and then in 0.8% Tween 80 with pH 7.5, with resultingconcentration 10%.

Forty female SKH-1 hairless mice were divided into control and testgroups, with 20 in each. The mice in both groups were treated topicallyonce with 5.12 μg of DMBA dissolved in 200 μl acetone per mouse toachieve tumor initiation. One week later (day 8), animals in the testgroup were applied topically with 200 μl iso-C15 per application permouse per day. The control group received 200 μl Tween 80 perapplication per mouse per day. Thirty minutes later, the animals in bothgroups were exposed to UVB (290–320 nm) radiation at a dose of 180mJ/cm² per day to achieve UVB radiation-induced tumor promotion. Theiso-C15 or vehicle treatments followed by UVB irradiation were performedtwice a week up to 30 weeks from the start of UVB exposure. The animalswere evaluated for tumor incidence at the end of 30 weeks.

Results:

The results assessed the protective effect of iso-C15 during the tumorpromotion stage. At the termination of the experiment, the animals inthe iso-C15-treated group showed a 40% reduction in tumor incidence. Theiso-C15-treated group also showed 90% reduction of tumor volumecomparing to the vehicle-treated. i.e. control, group.

II. Production Process of Specific Branched-Chain Fatty Acids

The present invention includes methods of making said specificbranched-chain fatty acids with anticancer activities.

The specific branched-chain fatty acids of the present invention can beisolated from natural resources occurring including, but not limited to,the organisms containing the specific branched-chain fatty acids, suchas animal fats or phytol of green plants.

The specific branched-chain fatty acids of the present invention canalso be synthesized by chemical or biological methods. The classicalKolbe's synthesis methods of branched-chain fatty acids are well knownand a specific example of a method for electrosynthesis of13-methyltetradecanoic acid is provided in the example below. Thebiosynthesis methods for making the specific branched-chain fatty acidsof the present invention are fermentation or incubation processes usingspecific bacteria strains containing a high percentage of specificbranched-chain fatty acids in their cellular lipids. A process formaking a fermentation solution containing specific branched-chain fattyacids and having anticancer functions is also provided in the examplesbelow.

EXAMPLE 5 Electrolytical Synthesis of 13-Methyltetradecanoic Acid

13-methyltetradecanoic acid is synthesized electrolytically fromisovaleric acid and methyl hydrogen dodecanedioate in methanolicsolution, based on Kolbe electrolysis.

Dimethyl dodecanedioate was prepared from dodecanedioic acid byesterification with 5 parts v/w methanol containing 5% w/v concentratedsulfuric acid. The dimethyl ester, after purification by vacuumfractional distillation, was converted to the mono-ester using thetheoretical quantity of kalium hydroxide in anhydrous methanol. Themethyl hydrogen dodecan-1,12-dioate was purified by distillation.

The electrolytic coupling reaction was carried out with mono-ester and a2-fold molar excess of isovaleric acid dissolved in methanol containingsodium methoxide, using 2×10 cm² platinum electrodes. The reactionmixture was stirred and maintained at 50° C. by water cooling until thesolution became alkaline. Electrode polarity was reversed every 30 minto prevent the built-up of deposits on the electrode surfaces.

After electrolysis the reaction mixture was cooled to room temperatureand the by-products, dimethyl docosanedioate, which precipitated wasremoved by filtration. The filtrate was acidified with acetic acid andthe methanol removed by rotary evaporation under reduced pressure. Thecrude methyl 13-methyltetradecanoate was purified by fractionaldistillation. Finally the methyl ester was hydrolyzed by refluxing withexcess 10% w/v sodium hydroxide in ethanol/water (50:50, v/v). Aftercooling and acidification, the free acid was extracted with diethylether and purified by vacuum distillation.

EXAMPLE 6 Process for Making a Fermentation Solution Containing SpecificBranched-Chain Fatty Acids

A process for making a fermentation solution containing a highpercentage of specific branched-chain fatty acids is exemplified below.

The starter cultures grow in a slant agar medium for 24 hours first,then are inoculated onto the liquid medium in the culture flask andcultured on the incubator shaker for 24 hours. Next, the liquid culturesin the flask are inoculated onto a seeding tank at an inoculating rateof 0.1–0.5% (w/w). The cultures, after fermenting in the seeding tankfor 24 hours, are replaced onto production fermenters to ferment for 48hours, with aseptic airflow passing the mass. Generally, themagnification ration from seeding tank to production fermenter is aboutten. The incubation conditions are aeration rate of 1: 0.6–1.2(mass/air) v/v in, agitation speed of 180–260 rpm, and the temperatureof 28–38° C.

After the incubation is finished, the resulting culture solution isautoclaved at 100° C. for 30 minutes, and then the harvested solutioncan be packaged and autoclaved at 120° C. The resulting product is anoral nutritional liquid with anticancer and salutary functions for humanuse.

Alternative products can be obtained by a different procedure including,but not limited to, the method below. After the incubation is finished,an appropriate amount of hydrochloric acid can be added to the resultingculture solution to lower the pH to 3–4, and it is autoclaved at 100° C.for 30 minutes and the cooled solution is finally centrifuged. Theresulting supernatant, in which soy saponin is the predominantcomponent, can be used to manufacture a nutrient drink with varioustastes. Then the same volume of 95% aqueous ethyl alcohol and the samevolume of 2 N NaOH can be added to the resulting precipitate, which isthen agitated and then heated at 100° C. After cooling and centrifuging,while collecting the resulting supernatant for later use; the samevolume of 1 N HCl can be added to the remainder precipitate and heatedfor 5 minutes at 80° C. After cooling and centrifuging, the resultingsupernatant is collected. The two fractions of supernatants are combinedtogether and the pH adjusted to 9.0, to thereby obtain a concentratedoral nutritional liquid product containing various branched-chain fattyacids, and soy isoflavones such as saponin, daidzein, genistein, andother anticancer substances.

Another procedure is, after the incubation is completed, directlyatomize and dry the solution into a powder product, and encapsulate thepowder into capsules or make tablets.

The specific branched-chain fatty acids can be isolated from thefermented solution using well-known methods in the art for isolatingcellular fatty acid. The isolated active branched-chain fatty acids arereprocessed in various formulations. The formulations of the presentinvention comprise at least one specific branched-chain fatty acid orits pharmaceutically acceptable salt including sodium salt. They can becontained in an ampule for injection or transfusion, especially foradvance stage cancer patients. They may also be mixed with a carrier, ordiluted by a carrier, or enclosed or encapsulated by a digestiblecarrier in the form of a capsule, sachet, cachet, paper or othercontainer or by a disposable container such as an ampule. The carrier ordiluent may be a solid, semi-solid or liquid material, which serves as avehicle, excipient or medium for the active therapeutic substance.

In the process described above, soybean media are used. The componentsare listed by weight with water making up the reminder. Proper amountsof trace elements necessary for the human body in addition to saidnutritional components are added.

Soybean medium Soybean 5–10% or soybean milk or bean cake (by soybeanwt.) 5–15% Yeast extract 0.02–0.5% or yeast powder 0.02–0.5% CaCO₃0.05–0.25% K₂HPO₄ 0.02–0.10% MgSO₄ 0.01–0.05% NaCl 0.01–0.04% Na₂MoO₄5.0–30 ppm ZnSO₄ 2.5–15 ppm CoCl₂ 5.0–20 ppm

In addition to the bacteria which have been identified as containing ahigh percentage of branched-chain fatty acids, such as the genusStenotrophomonas, Xanthomonas, Flavobacterium, Capnocytophaga,Altermonas, Cytophage, Bacillus, Chryseobacterium, Empdobacter,Aurebacterium, Sphinggobacterium, Staphylococcus, Azotobacter andPseudomonas, the bacteria of the present invention also include allother bacteria strains containing branched-chain fatty acids.

The products in the form of oral liquids, capsules, tablets orinjections, produced using said bacteria and media, with the process ofmaking of the present invention, have anticancer functions and othernutritional effects for human and animals.

III. Anticancer Function of Fermentation Solutions Containing SpecificBranched-Chain Fatty Acids

The said fermentation solutions are produced using specific bacteriastrains, which contain a high percentage of branched-chain fatty acids,and nutritive media, such as soybean media, and processes of the presentinvention. The fermentation solution contains various specificbranched-chain fatty acids with significant anticancer activity, andother nutritional composites from soybean or other media and bacteriametabolite. To demonstrate their anticancer function, the followinganimal experiments and clinical trials are presented. As an example ofsaid fermentation solutions, in the following experiments, thefermentation solution, named Q-can oral liquid, was used.

Q-can oral liquid was produced using Stenotrophomonas maltophilia strainQ-can as the production strain, while using the soybean medium above andthe process of making of the present invention. Parallel animalexperiments and clinical trials were also conducted using its atomizedcapsule product. The same conclusions were obtained as for Q-can oralliquid product.

The production strain, Stenotrophomonas maltophilia Q-can, has all theidentifying characteristics of the sample on deposit with American TypeCulture Collection, 10801 University Boulevard, Manassas, Va.20110-2209, and assigned ATCC 202105. The bacterial characteristics wasidentified by ATCC as following:

The cellular morphology is motile, non-sporing, Gram negative, andaerobic rods.

The colony morphology is the following: Colonies at 24 hours on ATCCmedium #3 (nutrient agar) were I, −90% circular (approximately 1 mm indiameter) with entire margins and convex elevation, rough surface,semi-translucent, light beige color: II, −10% small circular (<1 mm indiameter), entire margins, convex elevation, semi-translucent, smoothsurface and darker than I. Same characteristics were found when grown onATCC medium #18 (T-soy agar). #44 (BHI agar) and #260 (Sheep bloodagar). Both colonies were characterized and found to be the same.

The cellular fatty acid composition of Stenotrophomonas maltophiliaQ-can is the following:

Fatty Acid (% of total) straight-chain acid* branched-chain acid** 10:00.48 i11:0 3.21 14:0 3.14 i13:0 0.50 15:0 0.33 i15:0 39.34 16:0 5.52a15:0 7.44 16:1 ω9c 3.37 i15:1 1.02 16:1 ω7c 12.58 i16:0 0.88 hydroxyacid i17:0 3.68 3OH-10:0 0.12 i17:1 ω9c 5.27 3OH-i11:0 1.51 i19:0 0.333OH-i12:0 2.68 3OH-i13:0 3.57 2OH-13:0 0.29 *The number to the left ofthe colon refers to the number of carbon atoms, the number to rightrefers to the number of double bonds. **i = iso fatty acids, a = anteisofatty acids.

Since the fatty acid composition of bacteria is influenced bybiosynthesis conditions including temperature and pH, the data above maybe considered as a typical value.

The typical fatty acid contents in 500 ml of Q-can oral liquid are thefollowing:

straight-chain acid branched-chain acid 10:0 2.0–2.7 mg i11:0 11.2–15.4mg 12:0 2.9–4.0 mg i15:0 106.0–145.8 mg 14:0 13.0–17.7 mg i16:0 3.1–4.3mg 15:0 2.8–3.8 mg i17:0 12.4–17.0 mg 16:0 251.7–346.1 mg i19:0 2.2–3.0mg 17:0 2.9–4.0 mg a15:0 23.4–32.1 mg 18:0 75.6–104.0 mg i17:1 ω9c4.1–5.7mg 20:0 5.5–7.6 mg 12:1 ω8c 4.3–5.9 mg hydroxy acid 16:1 ω7c21.0–28.9 mg 3OH-i11:0 6.3–8.6 mg 18:1 ω9c 488.8–672.0 mg 3OH-12:012.0–16.5 mg 18:2 ω6c 825.9–1135.6 mg 3OH-i13:0 13.2–18.1 mg

A. Animal Studies

EXAMPLE 7 Acute Toxicity Test of Q-Can Oral Liquid

Materials and Methods:

Q-can oral liquid; ICR mice weighing 20.5–22.5 g.

Based on the preliminary test which could not determine 50% lethal dose(LD50), twenty ICR mice (half each sex), weighing 20.5–22.5 g infasting, were given intragastrically with the most endurable capacity of3 ml Q-can oral liquid per mouse for four times within 24 hours (6 am.,10 am., 4 p.m., and 10 p.m.)

Results:

It was observed that all the tested mice were less active five minutesafter every administration, and returned to normal about one hour later.Three mice suffered from diarrhea one or two days after theadministration, but none of tested mice died within the following sevendays. After the course of treatment, the tested mice were sacrificed anddissected. Visual observation showed no abnormality in internal organs.This limited test indicated that Q-can oral liquid had no toxic effects,even when large doses were taken acutely. Based on the conversion ofbody surface area, this dose corresponds to 4642 ml Q-can oral liquidper day for an adult weighing 70 kg.

EXAMPLE 8 Subacute Toxicity Test of Q-Can Oral Liquid

Materials and Methods:

Q-can oral liquid: Kunming mice weighing 22–24 g.

Twenty-four mice (half each sex) were randomly divided into a controlgroup and a test group, and were administrated intragastrically with thenormal saline in the control group and Q-can oral liquid in the testgroup at a dosage of 0.8 ml per day for 21 days. On the 22nd day, twomice randomly selected from each group were scarified; the paraffinsections of their viscera were made for microscopic examination.

Results:

No pathologic changes in internal organs were found by either visualobservation or observation under microscope. The remaining ten mice ineach group were further observed for seven additional days. No mice diedin this observation period. This test showed that intragastricaladministration of Q-can oral liquid for 21 consecutive days did notresult in toxicity or pathologic changes in mice.

EXAMPLE 9 Long-Term Toxicity Test of Q-can Oral Liquid

Material and Methods:

Q-can oral liquid; Forty male and forty female Spraque-Dawley ratsweighing 60±0.75 g were supplied by Sino-English Joint Ventured ShanghaiSipure-Bikai Experimental Animal Co. Ltd.

Eighty mice were randomly divided into four groups: a high-dose group(20 ml/kg Q-can oral liquid); a mid-dose group (10 ml/kg Q-can oralliquid); a low-dose group (5 ml/kg Q-can oral liquid) and a controlgroup (10 ml/kg normal saline). The samples were given intragastricallyonce a day for three months. Over the course of the experiment,behavior, appetite, gastrointestinal reaction and body weight of therats were recorded. The index of blood routine, blood platelet,electrocardiogram, liver function and renal function were measured. Thetested rats were sacrificed and dissected after three-monthadministration. Visual and pathological examinations were made for theirmain organs including heart, liver, spleen, lung, kidney, stomach,jejunum and brain.

Results:

Generally speaking, the tested rats were well, no abnormal behavior, nogastrointestinal reaction, good appetite. The curve of weight increaseof the test group was similar to the control group (p>0.05). Theelectrocardiograph examination result was normal. The hematology(including blood routine and blood platelet) was not statisticallydifferent between the test group and control group (p>0.05). The liverfunction (including ALT and TTT) and the renal function (including BUNand Cr) showed no obvious changes either (p>0.05). Although thecreatinine of the test group was a bit higher, it was still in thenormal range. The pathological section examination of the main organsshowed that the cell structure and histomorphology in the test groupwere not obviously different from those in the control group. It isconcluded that Q-can oral liquid can be used safely, based on the factthat continuous administration had no toxicity reactions.

EXAMPLE 10 Effects of Q-can Oral Liquid on Prolonging Life-Span of FruitFly Method

Fruit flies of both sexes were divided into control and test groups. Inmaking forage fed to flies, water was used in the control group, while2%, 10% and 20% concentrations of Q-can Oral liquid were used in thetest groups, respectively. The numbers of dead fruit flies were countedevery day till the last one died. The mean life-span (mls) and themaximum life-span (MLS) were calculated.

Results:

TABLE 10 Effects on mean and maximum life-span of fruit flies mls (d, M± SD) MLS (d, M ± SD) group male female male female control 31.9 ± 9.726.9 ± 9.6   40.0 ± 4.3 43.0 ± 4.9 (46) (47) [0] [0] Q-can 2% 30.8 ± 6.531.8 ± 9.5*  39.5 ± 2.1 63.5 ± 0.7 (45) (48) [0] [1] Q-can  38.9 ± 13.3*35.9 ± 12.4** 64.5 ± 2.1 61.0 ± 4.2 10% (45) (47) [5] [0] Q-can   50.8 ±15.8** 45.5 ± 14.6** 64.5 ± 2.1 64.5 ± 2.1 20% (44) (47) [12]  [4] ( )case number, [ ] residual number of flies, comparing with control, *p <0.025, **p < 0.001 Q-can Oral Liquid significantly increased the meanand maximum life-span of fruit flies, suggesting its antiaging function.

EXAMPLE 11 Effects of Q-can Oral Liquid on Hepatic and CerebralLipoperoxide Method

Forty Balb/C mice of both sexes were randomly divided into control andthree test groups. The mice in the test groups were supplied with Q-canOral Liquid instead of drinking water for four weeks, and then werestarved for 24 hours. The malondiadehyde (MDA) contents were measured todetermine the hepatic and cerebral lipoperoxide (LPO) levels.

Results:

TABLE 11 Effect of Q-can on hepatic and cerebral LPO level (MDA nmol/g,X ± SD) male female group case liver brain liver brain control 10 75.8 ±5.50  106.8 ± 2.67  74.8 ± 4.93  108.8 ± 4.10   Q-can 30% 10 56.3 ±2.20* 83.0 ± 4.83* 55.0 ± 3.13* 87.0 ± 5.13*  Q-can 20% 10  63.2 ±2.30*°  92.5 ± 3.27*°  60.8 ± 3.07*° 94.0 ± 3.39*° Q-can 10% 10 62.2 ±3.33* 96.7 ± 3.83* 61.0 ± 2.27* 98.8 ± 2.50*° comparing with control: *p< 0.001, comparing between Q-can 20% and 30%: °p < 0.01 Q-can OralLiquid significantly decreased the hepatic and cerebral LPO levels,suggesting its antiaging function.

EXAMPLE 12 Enhancement of the Effectiveness of Chemotherapeutic Drugs

Materials and Methods:

Q-can oral liquid; mouse liver cancer HAC cell line; male Kunming miceweighing 20–25 g; commercial cyclophosphamide (CP).

Forty mice were randomly divided into 5 groups. In three test groups,36%, 60% and 100% Q-can oral liquids (diluted with water) were given,respectively, while water was given in both control groups. On the 8thday, 0.2 ml HAC cancer cell suspensions (10⁷/ml) were injected into theabdominal cavities of each mouse under aseptic condition. On the 1st,3rd and 5th days after injection. CP (50 mg/kg) was injected i.p. intothe mice in all test groups and positive control group. From the 9th dayafter injection, normal feeding was resumed as before the test. Date ofdeath of each mouse was recorded, and the average life span and the rateof increase in life span were calculated. The rate of increase in lifespan (ILS %) is defined as following:

${\frac{{{Life}\mspace{14mu}{span}\mspace{14mu}{of}\mspace{14mu}{test}\mspace{14mu}{group}} - {{life}\mspace{14mu}{span}\mspace{14mu}{of}\mspace{14mu}{control}\mspace{11mu}{group}}}{{Life}\mspace{14mu}{span}\mspace{14mu}{of}\mspace{14mu}{control}\mspace{14mu}{group}} \times 100}\%$

Results:

The results below (Table 12) showed the enhancement of anticancer effectof chemotherapeutic drug, CP, by combining treatment with Q-can oralliquid. The average life span of mice in the control group (withoutdrug) was only 10.63±1.03 days, while that in the positive control group(only taking CP) was 13.06±3.03 days, with ILS of 22.86%. Thecombination of Q-can oral liquid (with dosage of 60% and 100%) and CPincreased the effectiveness of CP, as evidenced by increased average ILSand increased numbers of mice that survived over 17 days (60%prolonged). Therefore the ILS rate by CP treatment has been increased56.78% and 143.86% by combination with 60% and 100% Q-can oral liquid,respectively. The difference was statistically significant.

TABLE 12 Q-can oral liquid enhanced the effectiveness of CP for livercancer group drug life span mice No (>17 d) prolonged days ILS % p* 1 —10.63 ± 1.03 0 — — 2 36% Q-can + CP 12.38 ± 2.37 1 1.75 ± 2.05 16.46<0.10 3 60% Q-can + CP 14.44 ± 3.54 2 3.94 ± 2.87 35.84 <0.02 4 100%Q-can + CP 16.56 ± 3.96 6 5.94 ± 2.96 55.75 <0.01 5 CP 13.06 ± 3.03 12.44 ± 2.58 22.86 <0.05 *compared with Group 1 (control group)

EXAMPLE 13 Tumor Inhibition Effects on Mouse Lewis Lung Tumor

Materials and Methods:

Concentrated Q-can oral liquid (containing specific branched-chain fattyacids 3.6 mg/ml); Female F1 mice (C57/B1 and DBA/2) weighing 18–22 g;Lewis mouse lung tumor.

The mice of the test group were administrated with concentrated Q-canoral liquid at a daily dose of 36 mg specific branched-chain fatty acidsper kg weight for 10 days before transplantation of Lewis mouse lungtumor. All the mice were transplanted subcutaneously in the subaxillaryregion with a piece of Lewis tumor of approximately 2 mm in diameter.The treatments of intraperitoneal injection of chemotherapy drug Cytoxan(CTX) in 30 mg/kg were given once a day after transplantation over an 8day period for the positive control group. The treatments for the normalcontrol group were daily injection of normal saline for 8 days. Theadministration of concentrated Q-can oral liquid for the test group wasinitiated 10 days before tumor transplantation and continued for another8 days. Finally all the animals were sacrificed by spinal elongation.Tumors were excised and body and tumor weights were recorded.

Results:

The significant inhibition effect of Q-can oral liquid was shown in thetest data (Table 13.) Although the inhibition rate of the positivecontrol group (chemotherapy drug CTX i.p. injection) was higher than thetest group, it is noticed that viability of the positive control (70%,only 8-day period) was lower than the test group (100%, 18–28 days),implying toxicity of CTX. As oral administration is expected to be lesseffective than intraperitoneal injection, change in route ofadministration or increase in dosage should enhance the tumor inhibitoryrate of Q-can oral liquid.

TABLE 13 Effects of Q-can Oral Liquid on Mouse Lewis Lung CarcinomaXenograft Implanted into Subcutaneous Area of Nude Mice mice No. bodyweight tumor weight group dose route in./fi. in./fi. mean ± SD (g) TIR(%) p NS — i.p. 12/11 21.2/22.5 1.90 ± 0.96 — CTX 30 mg/kg i.p. 10/7 21.2/20.3 0.71 ± 0.36 62.6 <0.01 Q-can 36 mg/kg i.p. 10/10 20.9/21.91.11 ± 0.46 41.6 <0.05

EXAMPLE 14 Tumor Inhibition Effects on Human Gastric AdenocarcinomaSGC-7901 Xenografted into Nude Mice

Material and Methods:

Female Balb/c-nu/nu athymia mice, 6 weeks old, weighing 18–22 g, housedin specific pathogen free (SPF) condition throughout the course ofexperiment; concentrated Q-can oral liquid contains 3.6 mg/ml specificbranched-chain fatty acids; the drug used for positive control mitomycinC (MMC) was commercially available from Kyowa Hakko Kogyo Co., Ltd.,Japan.

Human gastric adenocarcinoma SGC-7901 xenograft was established andmaintained. For the experiment, the xenograft fragments of diameter ofabout 2 mm were inoculated subcutaneously into the right submaxillaryregions of nude mice. The animals were randomly divided into five groupsfive days after inoculation. NS and MMC (20 mg/kg) were given once a dayi.p. in normal and positive control groups, respectively, whileconcentrated Q-can oral liquid was given in test groups once a day p.o.,starting on the same day, at daily doses of 18, 36 and 72 mgbranched-chain fatty acids per kg weight, respectively, for 14 days.Experiment was terminated 20 days post-implantation, and mice weresacrificed by spinal elongation. Tumors were removed and the weights oftreated versus control tumors were compared. Inhibition rate wascalculated. The experiment was repeated once.

Results:

The results below indicated that Q-can oral liquid at effective doses of18, 36 and 72 mg/kg given p.o. once a day for 14 days after tumorinoculation offered antitumor activity against human gastricadenocarcinoma SGC-7901 xenograft with no marked toxicity. The tumorinhibition rate increased with dosage of oral administration. Theobvious shrinkage of the tumors was observed.

The results from the two tests (Test I and Test II are as follows):

TABLE 14 Effects of Q-can oral liquid on human gastric adenocarcinomaxenograft SGC-7901 implanted into subcutaneous areas of nude mice MiceBody wt. Tumor wt. Inhibition Group dosage route schedule In./Fi.In./Fi. X ± SD, g % p Test I NS — i.p. Qd × 14 12/12 21.9/23.3 1.17 ±0.45 — MMC 2.0 mg/kg i.p. Qd × 14 6/6 22.4/22.0 0.33 ± 0.24 71.49 <0.01Q-can 18 mg/kg p.o. Qd × 14 6/6 22.0/22.5 0.80 ± 0.42 31.19 >0.05 Q-can36 mg/kg p.o. Qd × 14 6/6 21.9/22.0 0.60 ± 0.45 48.23 <0.05 Q-can 72mg/kg p.o. Qd × 14 6/6 21.6/21.7 0.57 ± 0.35 51.28 <0.05 Test II NS —i.p. Qd × 14 12/12 21.6/23.5 1.15 ± 0.30 — MMC 2.0 mg/kg i.p. Qd × 146/6 21.1/21.1 0.30 ± 0.33 73.51 <0.01 Q-can 18 mg/kg p.o. Qd × 14 6/621.9/22.3 0.90 ± 0.59 21.58 >0.05 Q-can 36 mg/kg p.o. Qd × 14 6/622.0/21.6 0.66 ± 0.49 42.47 <0.05 Q-can 72 mg/kg p.o. Qd × 14 6/622.3/20.3 0.51 ± 0.37 55.45 <0.01

B. Clinical Trials

EXAMPLE 15 Clinical Trial on Effects of Q-can Oral Liquid onSupplementary Treatment of Cancer

Methods:

The clinical trial of the effects of Q-can oral liquid as asupplementary treatment of cancer was carried out by the cooperation offive hospitals in China. 333 cases of cancer patients were involved andwere randomly divided into two groups, chemotherapy and radiotherapy.The chemotherapy group included a control subgroup, which only tookchemotherapy and contained 131 cases, and a test subgroup, whichcombined chemotherapy with Q-can oral liquid and contained 136 cases.The types of cancer involved included gastric, hepatic, esophageal,colon, pulmonary and mammary cancers, which were distributed similarlyand comparably in the two subgroups (p>0.1). The radiotherapy groupincluded a control subgroup (radiotherapy only, 32 cases) and a testsubgroup (combination of radiotherapy and Q-can oral liquid, 34 cases).The types of cancers involved included nasopharyngeal and laryngealcancers, which were distributed similarly and comparably in the twosubgroups (p>0.1). Meanwhile, sex and age distribution of cancerpatients in test and control subgroups was comparable (p>0.1).

The dosage of Q-can oral liquid for the two test subgroups was 80 ml×2per day and it was given for two months.

The clinical observations and records were performed daily and filled inthe unified observation forms. The changes of the deficiency syndrome,symptoms, blood routine plus platelet counts, the toxic reaction ofchemotherapy or radiotherapy, and the side effects of Q-can oral liquidwere recorded weekly. The cardiac, hepatic and renal functions, theliving quality and the tumor size were examined or analyzed monthly. Theserum, albumin and globulin, cellular immune functions (lymphocytetransformation, NK cell and subgroup composition of T-lymphocytes) andthe humoral immunity were determined before and after the clinicaltrials.

Results:

A. Effects on Clinical Symptoms

Four classes of therapeutic effects on deficiency syndrome were definedas:

Significant effect—the symptoms of the deficiency syndrome disappearedor got a significant favorable turn at the end of therapy;

Improvement—the symptoms got a favorable turn at the end of therapy;

Stability—the symptoms remained unchanged;

No effect—the symptoms became worse at the end of therapy.

In the chemotherapy group, the effectiveness rate of the test subgroupwas 67.46% (significant effect plus improvement), which wassignificantly higher than that of the control subgroup (40.60%), p<0.01.In the radiotherapy group, the effectiveness rate of the test subgroupwas significantly higher than that of the control subgroup, p<0.05 basedon Ridit analysis.

TABLE 15 Symptom changes in the chemotherapy group mitigation stabilityaggra- symptom subgroup case case (%) case (%) vation p appetite Test 7249(68.06) 18(25.00) 5(6.94) <0.01 control 81 18(22.22) 33(40.74)30(37.04) weakness Test 90 56(62.22) 28(31.11)  6(16.67) <0.01 control70 13(13.57) 29(41.43) 28(40.00)

TABLE 16 Weight change in the chemotherapy group increase stabilitydecrease subgroup case case (%) case (%) case (%) p test 136 63(46.32)33(24.27) 40(29.41) <0.01 control 131 20(15.27) 35(26.72) 76(58.01)*increase and decrease were defined as more than 0.5 kg changes of bodyweight, and intermediate was stability.

B. Effect on Immune System

TABLE 17 Cellular immunity changes in the chemotherapy group Pre-treatPost-treat item subgroup case (X ± SD)% (X ± SD)% p LTT* test 65 55.95 ±8.02 56.28 ± 8.55 <0.01 control 75 55.85 ± 8.87  49.41 ± 12.21 CD₃ test30 43.53 ± 4.55 43.47 ± 5.10 <0.01 control 30 45.47 ± 3.56 38.57 ± 4.50CD₄ test 30 45.07 ± 4.60 43.10 ± 5.13 <0.01 control 30 42.60 ± 5.2038.27 ± 5.62 NK cell test 15  9.60 ± 5.11 12.00 ± 4.23 <0.01 control 1412.96 ± 4.31 10.80 ± 4.00 *LTT: Lymphocyte Transformation Test

TABLE 18 Humoral immunity changes in the chemotherapy/radiotherapygroups Item Pre-treat Post-treat (g/L) group subgroup case (X ± SD)% (X± SD)% p IgG chemotherapy test 71 10.90 ± 4.69  11.92 ± 5.06  <0.01control 75 11.90 ± 4.38  11.05 ± 4.99  IgA chemotherapy test 72 1.63 ±0.67 1.73 ± 1.32 <0.01 control 75 1.65 ± 0.76 1.38 ± 0.76 radiotherapytest 34 1.74 ± 1.31 2.39 ± 2.18 <0.01 control 31 2.07 ± 1.03 1.88 ± 0.80IgM chemotherapy test 71 1.24 ± 0.59 1.55 ± 1.05 <0.01 control 75 1.53 ±0.78 1.30 ± 0.73

The cellular and humoral immunity was enhanced in the test subgroup ofcombining chemotherapy and Q-can oral liquid. The concentration of IgAincreased in the test subgroup of combining radiotherapy and Q-can oralliquid.

C. Effects on Chemotherapeutic Toxic Reaction

TABLE 19 Effects on toxic reaction of blood system sub- Pre-treatPost-treat item group case (X ± SD) (X ± SD) p WBC test 30 4.74 ± 1.215.45 ± 0.86 <0.01 (× 10⁹) control 30 5.29 ± 0.85 4.45 ± 0.80 Neutrophiltest 30 3.20 ± 0.82 3.66 ± 0.69 <0.01 cell control 30 3.72 ± 0.58 3.09 ±0.45 Hb (g/L) test 30 94.63 ± 18.00 96.89 ± 16.08 <0.01 control 30103.67 ± 13.24  99.20 ± 11.63 platelet test 30 140.30 ± 4.88  160.03 ±4.36  <0.01 (× 10⁹/L) control 30 157.33 ± 3.52  145.53 ± 5.33 

The blood routine and platelet quantity in the test subgroup droppedless than those in the control subgroup. This indicated that Q-can oralliquid can prevent the hemogram decrease caused by chemotherapy.Meanwhile, Q-can oral liquid was effective on the patients whose WBC andHb were lower than normal before chemotherapy.

TABLE 20 Effects on hepatic function of the chemotherapy group SGPT(nmol/L, X + SD) subgroup case pre-treat post-treat p test 89 460.06 ±25.34  330.11 ± 245.01 <0.05 control 84 261.47 ± 191.23 284.00 ± 217.30

TABLE 21 Effects on serum protein of chemotherapy pre-treat post-treatitem subgroup case (g/L, X ± SD) (g/L, X ± SD) p total protein test 101 65.31 ± 10.01 67.47 ± 5.99 <0.01 control 103 65.64 ± 6.53 64.20 ± 6.07albumin test 107 38.78 ± 5.65 39.13 ± 5.26 <0.01 control 102 39.44 ±4.74 38.18 ± 5.24

SGPT decreased and serum total protein increased in the test subgroup ofcombining chemotherapy and Q-can oral liquid. The results showed thatQ-can oral liquid could alleviate the damage of hepatic functions causedby chemotherapy and promote protein synthesis, thus protecting theliver.

TABLE 22 Effects on renal functions of the chemotherapy group Blood ureanitrogen (nmol/L) Blood creatine (nmol/L) subgroup case Pre-treatpost-treat case Pre-treat Post-treat p test 111 5.13 ± 2.95 4.95 ± 1.33110 97.15 ± 30.64  97.99 ± 23.46 <0.01 control 100 4.26 ± 1.03 5.04 ±1.42  90 89.28 ± 22.13 107.08 ± 41.27

Blood urea nitrogen and creatine decreased in the test group, whichindicated that Q-can oral liquid could alleviate the damage of renalfunction caused by chemotherapy.

In summary, compared with the chemotherapy only treatment of 131 casesof cancer patients, the results of combinational treatment with Q-canoral liquid showed markedly enhanced therapeutical effects withstatistical significance. These effects included amelioration of thedeficiency syndrome, improvement of the appetite, weakness, livingquality and immune functions, mitigation of the degree of leucopenicaction induced by chemotherapy, alleviation of the low leukocyte countand the hemoglobin concentration which decreased after treatment, andprotection of the hepatic and the renal functions. In comparison withthe radiotherapy only treatment, the amelioration of the deficiencysyndrome and increase of the serum IgG level were found in cancerpatients, who were treated by combination of radiotherapy with Q-canoral liquid. Q-can oral liquid had no toxic effects on the blood, heart,liver and kidney. Thus, Q-can oral liquid can be used as a supplementarytherapeutic agent for cancer patients.

EXAMPLE 16 Clinical Observation for 35 Cases of American Prostate CancerPatients Treated by Q-can Oral Liquid

The effect of Q-can oral liquid on PSA levels was tested for 8–18 weeks(average 14 weeks) in two hospitals in the USA, where an integrativeapproach to treating prostate cancer was applied. Patients were not onradiotherapy, chemotherapy, or hormonal treatment during the recordingperiod and followed a customized nutritional protocol. At a daily dosageof 250 ml concentrated Q-can oral liquid (containing 300 mg specificbranched-chain fatty acids), assay of PSA level was made for allpatients. The average drop in PSA level was noted. It is also found thatdrops in PSA level of the patients who had higher pre-treat PSA levelwas more significant than those of the patients who had lower pre-treatPSA level.

TABLE 23 The effects of Q-can oral liquid on PSA level (mg/ml) CaseNumber Pre-treat (mean ± SD) Post-treat (mean ± SD) p 35 10.2 ± 10.727.45 ± 6.06 <0.01

EXAMPLE 17 Effects of Iso-C15 on Psoriasis Skin Disease

13-methyltetradecanoic acid (iso-C15) was prepared by dissolving in NaOHsolution and then in 0.8% Tween 80 with pH 7.5, with resultingconcentration 10%. The iso-C15 cream was prepared with liposometechnology.

Three psoriases patients topically applied iso-C15 cream on skin lesionsthree times per day for one month. The symptoms were obviously relieved(itching, flaking and red patches), and psoriases spots disappeared inone patient and 50% area reduced in the other two.

EXAMPLE 18 Industrial Process for Making Fermentation Liquid

This example describes one method of industrial production of Q-can oralliquid in a more detailed manner.

Medium composition is: soybean 40 kg (milling to milk and removingresidue), K₂HPO₄ 200 mg, CaCO₃, 200 g, yeast extract 160 g, MgSO₄ 80 g,NaCl 80 g, Na₂MoO₄ 10 ppm. ZnSO₄ 10 ppm, CoCl₂ 5 ppm, NaHNO₃ 2 ppm,soybean oil (as antifoam addition) 4 kg, and add water to 400 kgtotally.

The above media is put into a seeding tank and lead steam 120° C. for 30minutes, then cooled to 30° C. Onto the seeding tank are inoculated 3 kgliquid cultures, which were cultured on the incubator shaker at 30° C.for 24 hours. Fermentation proceeds in the seeding tank for 24 hours,30° C. temperature, 200 rpm agitation speed, and 1:1 (v/v min) aerationrate. After confirming no infection under microscope, it is thentransferred into a 10 ton production fermenter for 48 hours, wherecompared to that in the seeding tank before, the media is ten times inquantity and the same percentage of the composition, and the sameparameters of temperature, agitation speed and aeration rate are used.When fermentation is finished and no infection is confirmed undermicroscope, the temperature is increased to 100° C. to autoclave for 30minutes. The cooled solution can be packaged and the packaged fermentedsolution is again autoclaved at 118° C. for 45 minutes. This is asemi-finished product waiting for quality inspection and final packageas the Q-can oral liquid product.

Every description in the above specification of a numerical range and ofa genus is intended to inherently include a description of all possiblevalues and subranges within the range, and all possible species andsubgenuses within the genus, respectively.

The disclosures of U.S. application Ser. No. 09/173,681 filed Oct. 16,1998, and of Provisional Patent Application No. 60/081,712, filed Apr.14, 1998, are hereby incorporated by reference.

1. A method of making a terminally methyl-branched iso- or anteiso-fattyacid, or a mixture of said fatty acids, which comprises culturing abacteria strain containing said fatty acid(s) to form a fermentationsolution containing said fatty acid(s), and then isolating said fattyacid(s), from the fermentation solution, wherein the bacteria strain isfrom the genus Stenotrophomonas.
 2. The method of claim 1, wherein theculture medium comprises a soybean medium.
 3. The method of claim 2,wherein the soybean medium has the following formula: Soybean 5–10% orsoybean milk or bean cake (by soybean wt.) 5–15% Yeast extract 0.02–0.5%or yeast powder 0.02–0.5% CaCO₃ 0.05–0.25% K₂HPO₄ 0.02–0.10% MgSO₄0.01–0.05% NaCl 0.01–0.04% Na₂MoO₄ 5.0–30 ppm ZnSO₄ 2.5–15 ppm CoCl₂5.0–20 ppm.


4. The method of claim 1, wherein the bacterial strain isStenotrophomonas maltophilia.
 5. The method of claim 4, wherein saidbacterial strain is assigned ATCC
 202105. 6. A method of making afermentation solution containing at least one terminally methyl-branchediso- or anteiso-fatty acid, which comprises culturing a bacteria straincontaining said fatty acid in a nutritive medium to form a fermentationsolution containing said fatty acid, wherein the bacteria strain is fromthe genus Stenotrophomonas.
 7. The method of claim 6, wherein thenutritive medium comprising a soybean medium.
 8. The method of claim 7,wherein the soybean medium has the following formula: Soybean 5–10% orsoybean milk or bean cake (by soybean wt.) 5–15% Yeast extract 0.02–0.5%or yeast powder 0.02–0.5% CaCO₃ 0.05–0.25% K₂HPO₄ 0.02–0.10% MgSO₄0.01–0.05% NaCl 0.01–0.04% Na₂MoO₄ 5.0–30 ppm ZnSO₄ 2.5–15 ppm CoCl₂5.0–20 ppm.


9. The method of claim 6, wherein the bacterial strain isStenotrophomonas maltophilia.
 10. The method of claim 9, wherein saidbacterial strain is assigned ATCC
 202105. 11. A product made by themethod of claim
 6. 12. A product made by the method of claim
 7. 13. Aproduct made by the method of claim
 8. 14. A product made by the methodof claim
 9. 15. A product made by the method of claim
 10. 16. Theproduct of claim 11, which is in the form of a liquid, powder, capsule,tablet, injection, or encapsulated with liposome, or topically appliedin the form of a cream, ointment, or lotion.
 17. The product of claim12, which is in the form of a liquid, powder, capsule, tablet,injection, or encapsulated with liposome, or topically applied in theform of a cream, ointment, or lotion.
 18. The product of claim 13, whichis in the form of a liquid, powder, capsule, tablet, injection, orencapsulated with liposome, or topically applied in the form of a cream,ointment, or lotion.
 19. The product of claim 14, which is in the formof a liquid, powder, capsule, tablet, injection, or encapsulated withliposome, or topically applied in the form of a cream, ointment, orlotion.
 20. The product of claim 15, which is in the form of a liquid,powder, capsule, tablet, injection, or encapsulated with liposome, ortopically applied in the form of a cream, ointment, or lotion.